Alpha-actinin associates with polycystin-2 and regulates its channel activity

QIANG LI, NICOLAS MONTALBETTI, PATRICK Y. SHEN, XIAO-QING DAI, CHRISTOPHER I. CHEESEMAN, EDWARD KARPINSKI, GUANQING WU, HORACIO F. CANTIELLO AND XING-ZHEN CHEN

HUMAN MOLECULAR GENETICS

Año: 2005 p. 1587 - 1603

0964-6906

Polycystin-2 (PC2) is the product of the PKD2 gene, which is mutated in 10–15% patients of autosomal dominant polycystic kidney disease (ADPKD). PC2 is an integral transmembrane protein and acts as a calciumpermeable cation channel. The functional modulation of this channel by other protein partners remains largely unknown. In the present study, using a yeast two-hybrid approach, we discovered that both intracellular N- and C-termini of PC2 associate with a-actinins, actin-binding and actin-bundling proteins important in cytoskeleton organization, cell adhesion, proliferation and migration. The PC2-a-actinin association was confirmed by in vitro glutathione S-transferase pull-down and dot blot overlay assays. In addition, the in vivo interaction between endogenous PC2 and a-actinins was demonstrated by co-immunoprecipitation in human embryonic kidney 293 and Madin-Darby canine kidney (MDCK) cells, rat kidney and heart tissues and human syncytiotrophoblast (hST) apical membrane vesicles. Immunofluorescence experiments showed that PC2 and a-actinin were partially co-localized in epithelial MDCK and inner medullary collecting duct cells, NIH 3T3 fibroblasts and hST vesicles. We studied the functional modulation of PC2 by a-actinin in a lipid bilayer electrophysiology system using in vitro translated PC2 and found that a-actinin substantially stimulated the channel activity of reconstituted PC2. A similar stimulatory effect of a-actinin on PC2 was also observed when hST vesicles were reconstituted in lipid bilayer. Thus, physical and functional interactions between PC2 and a-actinin may play an important role in abnormal cell adhesion, proliferation and migration observed in ADPKD.PKD2 gene, which is mutated in 10–15% patients of autosomal dominant polycystic kidney disease (ADPKD). PC2 is an integral transmembrane protein and acts as a calciumpermeable cation channel. The functional modulation of this channel by other protein partners remains largely unknown. In the present study, using a yeast two-hybrid approach, we discovered that both intracellular N- and C-termini of PC2 associate with a-actinins, actin-binding and actin-bundling proteins important in cytoskeleton organization, cell adhesion, proliferation and migration. The PC2-a-actinin association was confirmed by in vitro glutathione S-transferase pull-down and dot blot overlay assays. In addition, the in vivo interaction between endogenous PC2 and a-actinins was demonstrated by co-immunoprecipitation in human embryonic kidney 293 and Madin-Darby canine kidney (MDCK) cells, rat kidney and heart tissues and human syncytiotrophoblast (hST) apical membrane vesicles. Immunofluorescence experiments showed that PC2 and a-actinin were partially co-localized in epithelial MDCK and inner medullary collecting duct cells, NIH 3T3 fibroblasts and hST vesicles. We studied the functional modulation of PC2 by a-actinin in a lipid bilayer electrophysiology system using in vitro translated PC2 and found that a-actinin substantially stimulated the channel activity of reconstituted PC2. A similar stimulatory effect of a-actinin on PC2 was also observed when hST vesicles were reconstituted in lipid bilayer. Thus, physical and functional interactions between PC2 and a-actinin may play an important role in abnormal cell adhesion, proliferation and migration observed in ADPKD.